Staphylococcus aureus ATCC 25923) and Gram negative (Escherichia coli ATCC 25922) bacteria.Two polysaccharides (cellulose and chitosan) and polyurethane dissolved in 1-ethyl-3-methylimidazolium chloride represented the matrix for the obtainment of new composite formulations comprised of lignin, ferrite–lignin hybrid and ketoconazole. The mechanical performances (Young’s modulus and compressive strength) increased with the filler addition. The nature of the filler used in the studied formulations influenced both bioadhesion and mucoadhesion parameters. It was found that the incorporation of lignin and ferrite–lignin hybrid into the matrix has influenced the in vitro rate of ketoconazole release, which is described by the Korsmeyer–Peppas model. All materials exhibited activity against Gram positive ( Synthesis of multifunctional materials has received much attention in the last decades due to their improved attributes. In spite of their lower properties as compared to synthetic ones, the natural polymers represent one of the most promising options towards a green and sustainable future.Cellulose and chitin are most abundant natural carbohydrate polymers, having a high degradable and recyclable potential. Cellulose (Cel) is the major component of biomass. It presents excellent mechanical and thermal resistance and is widely used for the production of papers, fibers, textile fabrics, explosives, plastics or composite materials. Cellulose is a linear polymer consisting of carbon, hydrogen and oxygen in the form of β-d-glucopyranose units linked by β-1,4-glycosidic bonds.Chitosan (CS) and its derivatives are among the promising materials for applications in waste and water treatment, agriculture, cosmetic, food or in medicine ,2,3 becaPolyurethanes (PU) have numerous applications, such as biomedical, textile, automotive, paintings, adhesives or coatings ,7, due tLignin (L) is an aromatic polymer consisting of monolignols of substituted phenylpropanoid alcohols. It derives mainly from lignocellulosic biomass that renders its structure directly dependent on the source and delignification method. Lignin exhibits low toxicity and, therefore, the lignin valorization depends on the development of new approaches to overcome challenges that mitigate its conversion relative to potential medical applications ,12,13. O3O4-LNPs renders them promising for cancer therapy and diagnosis, such as magnetic targeting and magnetic resonance imaging. The cytotoxicity assays of other authors [According to P. Figuerido et al. , lignin  authors  revealed authors .Ketoconazole (KK) was selected as a model drug and added into polysaccharides–polyurethane matrix as an antifungal agent, while keeping in mind its recommendations for the treatment of skin, hair and mucosa infections. The resulting material was characterized by mechanical tests and structural investigations. Furthermore, the drug release capacity as well as antimicrobial activity was evaluated.This work evidences the potential of polysaccharides as matrix for drug delivery and also a promising method to valorize lignin and its hybrids for controlled release applications.3)2·6H2O and ferric nitrate Fe(NO3)3·9H2O at 500 °C) were also added to matrix in order to assess their potential for controlled release applications.Cellulose , chitosan, 1-ethyl-3-methylimidazolium chloride and ketoconazole were purchased from Sigma-Aldrich and used without further purification. The polyurethane was synthesized according to Spiridon et al. . OrganosCellulose (1g), chitosan (0.25 g) and polyurethane (0.25 g) were dissolved in 10 g of 1-ethyl-3-methylimidazolium chloride at 100 °C under stirring for 6 h. Ketoconazole (0.025 g), lignin or cobalt ferrite lignin (0.05 g) were added into Cel-CS-PU matrix and various formulations were obtained . Composia—initial amount of KK; mb—amount of KK found in washings .The amount of KK released by washing was analyzed spectrophotometrically at λmax value of 254 nm at room temperature. The load KK was calculated according to the following equation:The highest KK load was recorded for Cel-CS-PU-KK (96.84%), followed by Cel-CS-PU-LH-KK (96.12%) and Cel-CS-PU-L-KK (95.65%).Bruker FTIR Spectrophotometer Vertex 70  equipped with an attenuated total reflection (ATR) device was used to evidence the interactions between components of the composites. All samples were acquired using a diamond crystal with ZnSe focusing element at room temperature.−1. The compressive stress σ (Equation (1)) and the strain ε (Equation (2)) were calculated according to the following equations:σ denotes normal stress (N/m2); F denotes normal force acting perpendicular to the area (N); A denotes the area of the plate (m2); Δl denotes the change in length (m); ol denotes initial length (m).The mechanical measurements were carried out on swollen samples, at room temperature, using a Shimadzu Testing Machine  equipped with a 500 N load cell for compression. The samples were cut into plates with a thickness, width and height of about 10 mm, 10 mm and 3 mm, respectively. A pre-load of 0.1 N was applied before the initiation of each test to ensure complete contact between the sample and the plates of the analyzer. The compression experiments were conducted at a cross-head speed of 1 mm minThe compressive Young’s modulus of all materials was determined from the slope of the linear part of the stress−strain curves in accordance with the procedure already reported for other bio-based hydrogels. Three different samples were tested for each composition and the values of the modulus of elasticity were expressed as an average value ± SD.The lyophilized samples were immersed in phosphate buffer saline (PBS) for 24 h to reach saturation prior their testing in a wet state. An initial compressive force of 0.1 N was applied in order to ensure a complete contact between the surface of the plate and that of the sample. The setup of the test and the calculus for the elastic modulus were performed in accordance with the earlier reported procedure .The tests were performed using a TA.XT Plus texture analyzer . The maximum force of the detachment and the work of adhesion (the area under the force/distance curve) were determined by using the analyzer software . The matThe morphology of materials was evaluated with a scanning electron microscope (SEM—Quanta 200 instrument equipped with an energy-dispersive X-ray spectroscopy (EDX) module, low vacuum mode working) without sputter coating by conducting matter.max value of 254 nm at room temperature. The concentrations were estimated based on the calibration curves drawn at the same wavelength. Phosphate buffer saline was used as blank for baseline correction. All tests were performed in triplicate and the results are expressed as mean ± standard deviation.In vitro release of ketoconazole from all formulations was performed using phosphate buffer saline (PBS) at 37 ± 0.5 °C and pH value of 7.4. Briefly, a weighed amount of materials were placed in a flask with PBS and aliquots of 0.5 mL of the supernatant were removed at different time intervals and diluted to a total volume of 3 mL prior to examination. The amount taken was replaced with fresh PBS in order to maintain the sink conditions. The released ketoconazole was analyzed spectrophotometrically at λStaphylococcus aureus 25923, Escherichia coli 25922 and Candida albicans and ATCC 10231 strains in peptone saline with a turbidity of 1° McFarland. A suspension of approximately 1500 UFC (colony-forming units/mL) was obtained by dilution. The surfaces of the tested materials and of the control sample were contaminated with 100 μL ATCC strain . The inoculate was extracted using a sterile swab soaked in peptone saline and has seeded on the surface of the specific environment after 24 h. The plates were incubated at 37 ± 1 °C for 24 h. The colonies were counted and compared to the control sample.Suspensions were prepared from −1, C–N bond to 1490 cm−1, N–H to 1571 cm−1 and C=O to 1645 cm−1. The signal from 860 cm−1 is generally attributed to the C-O-C bonds of the glucopyranose nucleus in polysaccharides.ATR-FTIR spectra  proved t−1, which is associated with the amount of the crystalline structure of cellulose and 899 cm−1  bond in cellulose). The hydrogen bond intensity (HBI) depends upon the crystallinity as well as the amount of bounded water. This index is represented by the absorption ratio at 3400 cm−1 (H-bonds between molecules) and 1320 cm−1 (CH rocking vibration of the glucose ring).The spectra are useful for estimating some structural parameters for the tested materials . Lateral−1 [Total crystalline index (TCI) is calculated as a ratio between absorbances at 1376 and 2902 cm−1 .Being an amorphous polymer, lignin’s addition into the matrix decreased the crystallinity degree of the material (TCI). The increase in HBI value by the addition of lignin suggests strong interactions between the matrix components. The addition of ketoconazole decreased the lateral ordering of polymeric matrix.Mechanical behavior is an important factor for the materials that could have potential applications for tissue engineering. Herein, a uniaxial compression test was performed in order to ascertain the effect of the added fillers  on the resulting formulations. The typical stress-strain compression curves are presented in −1. The insets of Typical compression stress-strain curves for Cel-CS-PU-based composites obtained by applying a normal force of 100 N under a displacement rate of 1 mm × minSwelling ratio of all formulations has been evaluated and it is well correlated with mechanical parameters. Swelling ratio displayed the following order: Cel-CS-PU (56.92%) > Cel-CS-PU-L (52.9%) > Cel-CS-PU-LH-KK (48.91%) > Cel-CS-PU-KK (48.03%) > Cel-CS-PU-L-KK (23.99%) > Cel-CS-PU-LH (19.96%).The morphological features of different formulations are shown in The nature of the filler used in formulations influenced both bioadhesion and mucoadhesion parameters. In Mucoadhesion of all formulations was assessed in the stomach at pH 2.6 at 37 °C. The tests performed with porcine stomach tissue evidenced an increase in the force of detachment when lignin and its hybrid was added into Cel-CS-PU matrix, suggesting the ability of matrix components to establish various types interactions (numerous hydrogen bonds and electrostatic interactions) with mucus . A good Other authors  have preDue to the fact that the controlled and slow release of drug systems have gained more attention as compared to other drug delivery systems, the evaluation of in vitro drug release performance was carried out in PBS solution to compare the release profile of KK from different formulations. This was affected by the interactions between the materials’ components and the fungal agent, as well as by the diffusion kinetics in the PBS medium. Thus, the drug release rate for the first 200 min was about 15% for Cel-CS-PU-KK and Cel-CS-PU-K-L and about 16.4% for Cel-CS-PU-LH-KK. The same trend was maintained after 500 min (about 49% vs. 56.2%).2/g) as compared with that of lignin (118.34 m2/g).According to The Korsmeyer–Peppas model has described the release of KK from all formulations.n . Moreover, C. Albicans was more susceptible in comparison with E. coli and S. aureus after addition of ketoconazole in polymeric matrix.The addition of lignin into the polymeric matrix increased the inhibition rate of Novel composites based on polysaccharides (cellulose and chitosan) and polyurethane dissolved in 1-ethyl-3-methylimidazolium chloride were obtained. Mechanical characterization evidenced that the Young’s modulus and compressive strength increased with the addition of lignin, ferrite–lignin hybrid or ketoconazole. This improvement is well-correlated with the increased cross-linking among components in more hydrogen bonds and confirmed by the HBI values.Staphylococcus aureus 25923 and Escherichia coli 25922) and yeast . It also recorded the highest value for both bioadhesiveness and mucoadhesiveness.The incorporation of lignin and ferrite–lignin hybrid into Cel-CS-PU-KK system has influenced the release profile of KK, which is described by Korsmeyer–Peppas model. The mechanism of drug release was controlled by the Fickian diffusion. Cel-CS-PU-L-KK exhibited the strongest antimicrobial activity, but all materials presented effective inhibition of the bacteria (All obtained data provide information on the suitability of polysaccharides as a matrix for drug delivery and as a promising method to valorize lignin and its hybrids in controlled release applications.


The aim of this study was to identify serum miRNAs that discriminate early gastric cancer (EGC) samples from non-cancer controls using a large cohort.®) designed to detect 2565 miRNA sequences. Diagnostic models were constructed using the levels of several miRNAs in the discovery set, and the diagnostic performance of the model was evaluated in the validation set.This retrospective case–control study included 1417 serum samples from patients with EGC  and 1417 age- and gender-matched non-cancer controls. The samples were randomly assigned to discovery and validation sets and the miRNA expression profiles of whole serum samples were comprehensively evaluated using a highly sensitive DNA chip . In the discovery set, a receiver operating characteristic curve analysis of the EGC index revealed that the area under the curve (AUC) was 0.996 with a sensitivity of 0.983 and a specificity of 0.977. In the validation set, the AUC for the EGC index was 0.998 with a sensitivity of 0.996 and a specificity of 0.953.A novel combination of four serum miRNAs could be a useful non-invasive diagnostic biomarker to detect EGC with high accuracy. A multicenter prospective study is ongoing to confirm the present observations.The online version contains supplementary material available at 10.1007/s10120-021-01161-0. Gastric cancer is the second most common malignancy and has a high mortality rate worldwide . AlthougThe updated version of the Japanese Guidelines for Gastric Cancer Screening recommends radiographic and endoscopic screening as effective tools to detect EGC ; howeverSeveral studies have investigated the role of circulating microRNAs (miRNAs), non-coding RNAs composed of 17–25 nucleotides, as diagnostic biomarkers of cancer . MiRNAs Our study group launched a national project in Japan entitled “Development and Diagnostic Technology for Detection of miRNA in Body Fluids”. This project includes a comprehensive characterization of the serum miRNA profiles of 13 types of human cancers, including EGC, in more than 10,000 patients using the same platform and technology. This aim of our current study was to develop a model to differentiate between EGC patients and non-cancer controls using the expression levels of serum miRNAs.This case–control study included 1417 serum samples from consecutive patients with initial EGC and without any other cancers at the National Cancer Center Hospital  between 2008 and 2012. All of the cancers were histologically proven and treated by either endoscopic resection or gastrectomy with lymph node dissection, according to the Japanese Gastric Cancer Treatment Guidelines . Serum sThe study was approved by the National Cancer Center Hospital Institutional Review Board , the Research Ethics Committee of the Medical Corporation Shintokai Yokohama Minoru Clinic (6019-18-3772), and the Ethics and Conflict of Interest Committee of the National Center for Geriatrics and Gerontology (754). Written informed consent for the use of serum samples for research purposes was obtained from each participant.total RNA was extracted from 300 µL of serum using 3D-Gene® RNA extraction reagent . A comprehensive miRNA microarray analysis was performed using a 3D-Gene® Human miRNA Oligo Chip version 21 . The miRNA signal values were normalized to the ratio of the average signal value of three internal control miRNAs  [The standardized method of serum miRNA analysis has been published previously . BrieflyiR-4463) . The valTo establish a diagnostic model, the serum miRNA profiles of EGC patients and non-cancer controls in the discovery set were compared using Fisher’s linear discriminant analysis with a greedy algorithm, as reported previously . The best-tests and categorical variables were compared using Pearson’s The diagnostic performance of the model was examined in the validation set. To confirm the robustness of the results, a subgroup analysis was performed for each non-cancer control group in the validation group. A subgroup analysis of EGC samples in the validation group was also performed for each pathological stage and histology classification. The clinicopathological information of EGC patients was collected from the hospital cancer registry. In patients with multiple EGCs, a main lesion was selected based on the depth of invasion and tumor size. The pathological stage was classified as IA, IB, or II, according to the guidelines of the International Union Against Cancer. The 7th edition clinical stage classification was used for patients who had a diagnosis of EGC in 2012, and the 6th edition was used for patients who had a diagnosis before 2012. The histology was classified into differentiated-type, undifferentiated type, or special-type, according to version 3 of the Japanese Classification of Gastric Cancer . ContinuP-values were reported as two-sided, and P < 0.05 was considered statistically significant.Fisher’s linear discriminant analysis was performed using R version 3.6.3  with compute.es package version 0.2-5, hash package version 2.2.6.1, MASS package version 7.3-51.5, mutoss package version 0.1-12, and pROC package version 1.16.2. Validation of internal control miRNAs was performed using the NormqPCR package in Bioconductor version 3.11. Principal component analyses and unsupervised clustering with Pearson's dissimilarity as a distance measure and Ward's method for linkage analysis were performed using Partek Genomics Suite 7.18.0723. All other statistical analyses were performed using IBM SPSS Statistics version 25 (IBM Japan). All This case–control study included 1417 serum samples from patients with EGC and 1417 age- and gender-matched controls. The samples were randomly divided into the discovery and validation sets , but was not significantly different to that of the five miRNA model . The EGC index was calculated as follows: (2.06054) × miR-4257 + (− 1.25451) × miR-6785-5p + (0.834875) × miR-187-5p + (− 1.07189) × miR-5739–4.4385. This index achieved a sensitivity of 0.983 and a specificity of 0.977. Figure Among the 2565 miRNAs examined, 414 had a normalized signal value exceeding 64 in more than 50% of samples in each group and were selected for further analysis. The three control miRNAs  were stably expressed in serum samples used in the present study (Supplementary Table 1). Table In the validation set, the AUC for the EGC index was 0.998 (95% C.I. 0.995–1.000), with a sensitivity of 0.996 (95% C.I. 0.991–1.000) and a specificity of 0.953 (95% C.I. 0.938–0.969) Fig. b. UnsupeIn the current study, we developed an EGC index to differentiate EGC from non-cancer controls based on the serum levels of four miRNAs . In the validation set, the EGC index demonstrated a sensitivity of 0.996 and a specificity of 0.953, with an AUC of 0.998. The sensitivities of the EGC index did not differ significantly among the clinical stages of EGC or between the three sets of non-cancer control samples.The updated version of the Japanese Guidelines for Gastric Cancer Screening recommends performing an upper gastrointestinal series and gastroscopy for population-based and opportunistic gastric cancer screenings . Some laHelicobacter pylori infection is a well-known risk factor for gastric cancer, and intestinal metaplasia is one of the most common pre-cancerous lesions that may lead to the disease [H. pylori and measuring the level of serum pepsinogens is a method of screening for EGC [ disease . A combi for EGC , 23. AltA variety of serum or plasma miRNAs are frequently upregulated or downregulated in gastric cancer . AlthougThe EGC index developed here includes four miRNAs: miR-4257, miR-6875-5p, miR-187-5p, and miR-5739. The serum levels of miR-4257 and miR-187-5p were higher in the EGC samples than in the non-cancer control samples, whereas the level of miR-6785-5p was lower in the EGC samples than in the control samples. Although the levels of miR-5739 were comparable in the EGC and control samples (AUC 0.463), AUC of the EGC index was higher when this miRNA was included than when it was excluded (Supplementary Table 2). Although the roles of these miRNAs in carcinogenesis remain unclear, some previous reports support the results of our study. Notably, miR-187-5p has already been described as a serum biomarker for the early detection of gastric cancer . In a stIt is important to analyze the diagnostic performance of the EGC index in advanced gastric cancer and other malignancies for further discrimination of EGC. Exploratory data were analyzed in ten patients with serum samples of Stage III locally advanced gastric cancer, 50 esophageal squamous cell carcinomas (ESCC), and 50 colorectal cancers (CRC). The AUCs of the EGC index in Stage III gastric cancer, ESCC, and CRC were 1.00, 0.640, and 0.440, respectively . Further discrimination models should be established in a large-scale cohort study.® in the diagnostic index of prostate cancer was reported previously by our study group [H. pylori infection, atrophic gastritis, and smoking, which could have influenced the levels of circulating miRNAs [Our current study has several limitations. First, the study was a retrospective analysis using archival samples, and an external validation cohort for patients with EGC was not available. Although the reproducibility of 3D-Genedy group , furtherg miRNAs , 34, becIn conclusion, the novel combination of serum miRNAs comprising miR-4257, miR-6785-5p, miR-187-5p, and miR 5739 could be a useful diagnostic biomarker to detect EGC with high accuracy.Supplementary file1 (PDF 1334 KB)Supplementary file2 (DOCX 17 KB)Supplementary file3 (JPG 1232 KB)Below is the link to the electronic supplementary material.


Results: The three populations were comparable in their baseline characteristics; however, the cryoballoon group comprised a higher percentage of patients with persistent AF (p = 0.05). The LAT rates were 3.7% in the iRF group (mean follow-up 22 ± 14 months), 0.7% in the PVAC group (mean follow-up 21 ± 14 months), and 4% in the cryoballoon group (mean follow-up 15 ± 8 months). The predominant mechanism of LAT was macro-reentrant tachycardia. Reconnection of at least one pulmonary vein was observed in 87% of the patients who underwent 3D mapping. No predictors for LAT occurrence were identified. Conclusion: The occurrence of LAT post PVI is rare; the predominant mechanism was macro-reentrant tachycardia. Reconnection of at least one pulmonary vein was observed in nearly all the LAT patients. In our retrospective analysis, the lowest rate of LAT was observed with the PVAC. No predictors for LAT occurrence were identified.Background: Left atrial tachycardias (LAT) are a well-known outcome of pulmonary vein isolation (PVI). Few data are available on whether the catheter used to perform PVI influences the incidence, as well as the characteristics of post PVI LAT. We present data on LAT following PVI by the following three ablation technologies: (1) phased multi-electrode radiofrequency catheter (PVAC), (2) irrigated single-tip catheter (iRF), and (3) cryoballoon ablation. Methods: Using a prospectively designed single-center database, we analyzed 650 patients  with paroxysmal ( Atrial fibrillation (AF) is the most widespread sustained arrhythmia  and a maA well-known outcome of PVI is the occurrence of left atrial tachycardias (LAT), which are often more symptomatic than the initial AF. It remains unclear if the technology used for PVI affects the occurrence rates and electrophysiological mechanisms of post PVI LAT. We sought to determine the occurrence rates, as well as mechanisms, of post PVI LAT after ablation, performed with the following three technical approaches: (1) single-tip irrigated RF catheter (iRF), (2) phased multipolar RF ablation catheter (PVAC), and (3) a cryoballoon system.We included a total of 650 patients who were referred to our center for an index PVI. Data were collected from a prospectively designed database, as well as our outpatient program. Patients were seen three months after PVI for an in-person visit. After 6 and 12 months, telephone interviews were performed. Further telephone interviews were scheduled in 6–12-month intervals. Additionally, patients were instructed to present to our department in case of symptomatic arrhythmias. Data collection was performed from 2011 to 2020. Exclusion criteria were defined as follows: severe valvular disease, prior surgical- or catheter-based PVI, prior linear ablations, inability to give informed consent, pregnancy, <18 years old.During the in-person visits and telephone interviews, patients were interrogated on arrhythmia occurrence. In the case of recurrence, ECG documentation was obtained. LATs within 3 months following PVI were discarded from the analysis.(1)iRF group: iRF ablation was guided by the NAVX system . A 3D model was created with a circular mapping catheter , and the acquired geometry was fused with the 3D reconstruction of the LA from computed tomography (CT). Ablation was performed with a 4 mm open-tip irrigated catheter . Antral point-by-point circumferential ablation around ipsilateral PVs, with a distance from 0.5 to 1.0 cm from the ostia, was performed. The power was set to 30 W and the temperature was limited to 43 °C. In case of persistent PV conduction, especially at the anterior ridge border of the lateral PVs, we increased the power to 35 W, to close conduction gaps. Complete electrical isolation was monitored and confirmed by the circular mapping catheter during sinus rhythm and differential pacing maneuvers. Ablation lesions were limited to circumferential PVI.(2)PVAC group: Ablation with the PVAC system was performed using the second-generation catheter, the PVAC GOLD . The PVAC was connected to the GENius (Medtronic Inc.) RF generator. The target temperature was 60 °C, with a maximum power of 8 W. Duty-cycled energy delivery was exclusively delivered in a 4:1 ratio, and the impulse duration was 60 s. Electrode pairs with insufficient tissue contact were selectively deactivated. After RF applications, the PVAC was rotated around the PV ostium, looking for the earliest pulmonary vein potential (PVP) to completely isolate the vein. Finally, the catheter electrodes were advanced in the PV demonstrating the entrance block; the exit block was not routinely checked for. No other catheters were used for touch-up lesions. No further ablations were performed.(3)Cryoballoon group: All cryoballoon patients were treated with the second-generation 28 mm cryoballoon . Occlusion of the PV was tested by a contrasting agent. In this cohort, 2 freezes were applied for 180 s per vein, irrespective of the achieved time to isolation (TTI). Additional freezes were applied if PVI was not achieved. Energy delivery was prematurely stopped in the case of inadequate temperature development, or if PVI could not be achieved within the first 90 s of the freeze. Ablation of the right PVs was performed under continuous stimulation of the phrenic nerve with a cycle length of 800 ms. Freezes were immediately stopped if temperatures fell below −60 °C, or if phrenic nerve capture was lost. Complete electrical isolation of the PVs was confirmed using the Achieve mapping catheter in sinus rhythm and during differential pacing maneuvers. At the end of the procedure, PVI was verified in sinus rhythm. No further linear ablations were performed and no supplementary ablation catheters were used.The catheter used for PVI was chosen at the operator’s discretion. Point-by-point RF ablation , ablation with the PVAC  system, as well as the cryoballoon  performed at our center have been described in detail in previous publications ,8,9,10. Mapping of LAT was performed using either NAVX  or CARTO  systems. In the more recent cases, multielectrode high-density catheters were used . The ablation catheter was either a 3.5 mm irrigated-tip ablation catheter  or a 4 mm irrigated-tip ablation catheter . In more recent cases, contact force sensing irrigated-tip catheters were used .A 3-dimensional activation and voltage map was acquired in all patients. The LATs were characterized upon their activation pattern, the presence of an “early meets late” region, and the differential pacing maneuvers . Peri-mitral flutter was usually treated by an anterior mitral isthmus line. Upon termination of LAT, conduction block of linear lesions was checked through differential pacing and activation re-mapping. Programmed atrial stimulation was performed in order to reinduce the atrial arrhythmia. Finally, in the case of PV reconnection, re-isolation was performed using the same RF catheter used for LAT ablation.p < 0.05 was considered significant.Statistical analysis was performed using SPSS . Continuous data were expressed as mean ± SD and compared between groups using a Mann–Whitney U test. Categorical data were summarized by their observed frequencies and percentages, and compared using cross tabulation and a chi-Square test. For all statistical tests, a value of A total of 650 patients  were included in the study. The three groups were comparable in baseline characteristics .All the patients were advised to stop all antiarrhythmic drugs 3 months after the index PVI. At the time of LAT ablation, the PVAC patient presented with flecainide. In the iRF group, one patient was on amiodarone, three were on dronedarone, and four were on flecainide/propafenone. In the cryoballoon group, three patients were on amiodarone at the time of LAT ablation.During a mean follow-up of 19 ± 8 months, 20 patients developed LAT. Three patients declined an electrophysiological study and opted for an electrical cardioversion. The diagnosis of LAT in these three patients was made based on the ECG, which was reviewed by at least two experienced electrophysiologists. In two patients, LAT terminated during or prior to 3D mapping; therefore, the LAT diagnosis was made through differential pacing.2; 2 PVs reconnected) developed peri-mitral macro-reentry during a mean follow-up of 21 ± 14 months. In the iRF group, 11 of the 300 patients included  developed LAT during a mean follow-up of 22 ± 14 months. In the cryoballoon group, eight LAT patients developed LAT over a mean follow-up of 15 ± 8 months .Of the 150 PVAC patients, 1 patient  ranged between 220 and 370 ms, with a mean of 252 ± 36 ms. Forty-five percent of the LAT patients had persistent AF at the time of the index PVI, and the mean left atrial volume (LAVI) was 40 ± 11 mL/mp = 0.76), CHA2DS2-Vasc (p = 0.1), male gender (p = 0.90), the presence of non-ischemic cardiomyopathy (NICM) (p = 0.39), the presence of ischemic cardiomyopathy (ICM) (p = 0.43), nor the left ventricular ejection fraction (LVEF) (p = 0.44) showed a significant correlation with the occurrence of LAT. Further, none of these characteristics reached statistical significance for predicting LAT occurrence in the different subgroups . For the cryoballoon group, the total freeze duration (p = 0.83), total number of freezes (p = 0.3), time to isolation (TTI) recorded in LSPV (p = 0.73), TTI recorded in RIPV (p= 0.09), and TTI recorded in RSPV (p = 0.63) showed no significant correlation with LAT occurrence. In the iRF and PVAC groups, the total RF durations also failed to reach statistical significance (p = 0.5 resp. p = 0.34). We performed a logistic univariate analysis to screen for predictors of LAT after PVI see . ConsideA direct comparison of the three different catheter technologies used for PVI ablation, in terms of their LAT rates, has, to the best of our knowledge, not yet been published. In the entire cohort, we were able to identify an occurrence of LAT of 3%. The lowest rate of LAT was observed with phased RF technology, followed by comparable LAT rates for point-by-point RF ablation and cryoballoon technology. No independent predictors of LAT occurrence were identified. Nearly all the patients with LAT showed mild-to-severe LA dilation, a well-known marker for advanced atrial electrical and structural remodeling . The majThe fibrosis due to advanced atrial remodeling, as well as supplementary ablation lesions and, especially, gaps in the lesion sets, serves as the arrhythmogenic substrate for re-entry. It is well established that the rate and mechanism of LAT after PVI varies with the lesion set chosen for AF ablation, as well as the lesion quality ,13,14. AOur rate of LAT in the iRF group is in line with previous publications, citing rates of 2.9% after isolation of the pulmonary veins alone ,16 or novel pulse field ablation technology reduces the rates of LAT remains to be determined. A comparative study in a pig model between conventional iRF and HPSD, with 90 W/4 s for PVI, showed HPSD to be superior to conventional iRF, in terms of lesion contiguity, transmurality, as well as uniformity . The firWe present a non-randomized study; the choice of ablation catheter was made at the operator’s discretion. Further, we performed a non-continuous follow-up and may have missed some asymptomatic LATs, although, in our experience, LATs are, in general, symptomatic. Further, the use of newer developments in point-by-point RF ablation, such as high-power, short-duration and contact force sensing catheters, may have affected the LAT occurrence rate in the iRF group.The occurrence of LAT after ablation of AF when limited to PVI only remains low, independent of the catheter technology or the energy source. The predominant mechanism of LAT was macro-reentrant tachycardia. In most patients with LAT, at least one PV was reconnected, which may play a role in LAT development. We did not find any predictors for LAT development.


Background and Purpose: The eosinophil-to-neutrophil ratio (ENR) was recently reported as a novel inflammatory marker in acute ischemic stroke (AIS). However, few studies reported the predictive value of ENR in AIS patients, especially for those with intravenous thrombolysis.Methods: Two hundred sixty-six AIS patients receiving intravenous thrombolysis were retrospectively recruited in this study and followed up for 3 months and 1 year. The Modified Rankin Scale (mRS) and the time of death were recorded. Poor outcome was defined as mRS 3–6. After excluding patients who were lost to follow-up, the remaining 250 patients were included in the 3-month prognosis analysis and the remaining 223 patients were included in the 1-year prognosis analysis.Results: ENR levels in the patients were lower than those in the healthy controls. The optimal cutoff values for the ability of ENR × 102 to predict 3-month poor outcome were 0.74 with 67.8% sensitivity and 77.3% specificity. Patients with ENR × 102 ≥ 0.74 have a lower baseline National Institutes of Health Stroke Scale (NIHSS) score . After multivariate adjustment, patients with ENR × 102 ≥ 0.74 were more likely to come to a better 3-month outcome . At the 1-year follow-up, the patients with ENR × 102 ≥ 0.74 showed a lower risk of mortality .Conclusions: A lower ENR is independently associated with a 3-month poor outcome and a 3-month and 1-year mortality in AIS patients treated with intravenous thrombolysis. Stroke is one of the leading causes of mortality and morbidity worldwide . IntraveA strong neuro-inflammatory response is characteristic of ischemic stroke . NeutropThe eosinophil-to-neutrophil ratio (ENR) is a novel biomarker that was reported to be associated with in-hospital mortality of patients with chronic obstructive pulmonary disease (COPD) . A recenThe data that support the findings of this study are available from the corresponding author on reasonable request.The detailed selection criteria of the study patients are displayed in Information of HCs was obtained from electronic examination reports. As for patients, the demographic data  and medical history  were obtained from medical records. National Institutes of Health Stroke Scale (NIHSS) scores on admission and stroke subtypes were evaluated by experienced clinicians. Blood samples were collected on 24 h of admission. ENR was calculated using eosinophil counts divided by neutrophil counts. At 3 months and 1 year after onset of AIS, the prognoses of patients were assessed through telephone follow-up by two clinicians.The etiology of AIS was classified on the basis of the Trial of Org 10,172 in Acute Stroke Treatment (TOAST) criteria: cardioembolic, atherosclerotic, small vessel or lacunar, and cryptogenic or others . Stroke via SPSS Statistics 24.0 software , R version 4.0.2 , and MedCalc Statistical Software version 15.2.2 . Continuous variables were expressed as medians and interquartile range while categorical variables were expressed as frequencies and percentage. The intergroup difference of continuous variables was compared using the Mann–Whitney U-test. The chi-square test was performed for categorical variables. ENR levels between HCs and AIS patients were compared before and after age and sex matching. In AIS patients, the optimal cutoff value of ENR to predict the 3-month poor outcome was determined using receiver operating characteristic (ROC) curve analyses, and then patients were divided into a high-ENR group (ENR ×102 ≥ 0.74) and a low-ENR group (ENR × 102 < 0.74). Univariate and multivariable logistic analyses were performed to estimate the association between ENR and AIS outcomes where variables with a p < 0.10 in univariate analysis were entered in the multivariable model. In addition, restricted cubic splines with four knots  were performed to further investigate the relationship between ENR and AIS outcomes. C-statistics, net reclassification index (NRI), and integrated discrimination improvement (IDI) were employed to assess the incremental predictive ability of ENR. For clinical practice, 1-year mortality was presented graphically using Kaplan–Meier curves and we used log-rank tests to compare survival between high-ENR group and low-ENR group. Cox regression was used to determine whether ENR is a significant predictor for 1-year mortality. Statistical significance was set at p < 0.05.Statistical analyses were performed 2 in AIS patients  compared to HCs. After matching of age and sex, ENR × 102 in AIS patients was still lower than that in HCs .Among all enrolled subjects, 266 were AIS patients and 2,196 were HCs. The characteristics of the AIS patients and the HCs are displayed in 2 that best distinguished the 3-month poor outcome were 0.74 with 67.8% sensitivity and 77.3% specificity; the area under the curve (AUC) was 0.738 . ENR had a better performance in discriminating patients at high risk and low risk of poor outcome than either eosinophil or neutrophil counts alone  (n = 154) and a low-ENR group (n = 96) according to the ENR cutoff values. The median ENR × 102 was 2.45 in the high-ENR group and 0.15 in the low-ENR group. A significant higher proportion of male, eosinophil count, and percentage of atherosclerotic stroke and a significant lower percentage of atrial fibrillation, neutrophil count, percentage of cardioembolic stroke, baseline NIHSS score, and 3-month mRS scores were observed in the high-ENR group  patients were lost to follow-up and the remaining 250 patients were included in the prognosis analysis. The optimal cutoff values of the ENR × 10= 0.726) . PatientNR group .p < 0.001) and mortality  was independently associated with 3-month function outcome  and mortality . Besides, the ENR as a continuous variable was also inversely associated with 3-month poor outcome . In a multivariate logistic regression model with restricted cubic splines, the elevated ENR level was associated with lower odds of 3-month poor outcome  had poor function outcome. In this cohort, patients in the high-ENR group had a decreased 3-month poor outcome , although no association was found between ENR and 1-year poor outcome when ENR was calculated as a continuous variable. Kaplan–Meier curves and the log-rank test indicated that patients in the high-ENR group had a lower incidence of mortality at the 1-year follow-up .At the 1-year follow-up, 43 (16.2%) patients were lost to follow-up and the remaining 223 patients were included in the prognosis analysis. Seventy-seven (34.5%) patients had a poor function outcome and 42 (18.8%) patients had died during the 1-year follow-up. After adjusting for age, current smoking, hypertension, atrial fibrillation, prior stroke, baseline NIHSS score, and stroke subtype, it is interesting that patients with ENR × 10ollow-up . MultivaA significantly decreased ENR level was observed in the AIS patients compared with the healthy controls. The ROC curve showed that ENR was a fair prognostic biomarker for 3-month poor outcome and had a higher predictive power than either eosinophil or neutrophil count alone. Patients with lower ENR levels were more likely to develop cardioembolic stroke and severe symptoms. In addition, the multivariate adjusted model and restricted cubic splines showed that elevated ENR levels were associated with a lower risk of poor 3-month function outcome. Furthermore, addition of ENR to the conventional model led to the improvement in the model's ability to predict a 3-month poor outcome. Our study also demonstrated that ENR is an independent predictor of 3-month and 1-year mortality in patients with AIS.in vitro and potentially in vivo neutrophil degranulation  patients finished the 3-month follow-up and 223 (83.8%) patients finished the 1-year follow-up. Furthermore, subjects of our study were selected from a single hospital so that selection bias may exist in our study.Our study shows that a lower ENR is independently associated with 3-month poor outcome and 3-month and 1-year mortality in AIS patients treated by r-tPA intravenous thrombolysis. Monitoring ENR at an early stage might be helpful for risk stratification and making therapeutic decisions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Third Affiliated Hospital of Wenzhou Medical University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SZ and GC conceptualized this work and supervised the study. HC, HH, CY, JR, JW, BG, WP, FS, XZ, TZ, JH, and YC acquired the data. HC, HH, CY, and JR performed the statistical analysis and interpreted the data. HC, HH, and CY prepared the manuscript. SZ, GC, HC, HH, CY, JR, JW, BG, WP, FS, XZ, TZ, JH, and YC revised the manuscript. All authors approved the protocol.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


For ROC analysis, the area under the curve (AUC) was 0.771 for Ret-He detecting ID and 0.845 for detecting IDA. The cut-off value for Ret-He to diagnose ID was 33.5 pg  and 31.6 pg  to diagnose IDA. Conclusions: The present study demonstrates Ret-He to be a screening marker for ID and IDA in children. Furthermore, Ret-He can be used as a single screening parameter for ID and IDA in children without considering other iron parameters. Economically, the use of Ret-He is highly relevant, as it can save one blood tube per patient and additional costs.Background: Iron deficiency (ID) is one of the most common nutritional deficiencies in children worldwide and may result in iron deficiency anemia (IDA). The reticulocyte hemoglobin equivalent (Ret-He) provides information about the current availability of iron in erythropoiesis. This study aims to examine the validation of Ret-He as a screening marker for ID and IDA in children. Methods: Blood samples were retrospectively obtained from medical records. Anemia was defined according to the definition provided by the World Health Organization (WHO) for children. ID was defined by transferrin saturation (TSAT) < 20% and ferritin < 100 ng/mL. Children were classified into four groups: IDA, non-anemia iron deficiency (NAID), control and others. Results: Out of 970 children, 332 (34.2%) had NAID and 278 (28.7%) presented with IDA. Analysis revealed that Ret-He significantly correlates with ferritin (rho = 0.41;  Iron deficiency (ID) is one of the most common nutritional deficiencies in children worldwide. In many cases, ID erythropoiesis results in iron deficiency anemia (IDA). In the pediatric population, the prevalence of anemia is highest (47.4%) in children of preschool age 5–15 years) 5 years ,3. . 3. Overall, anemia in children can be classified according to the World Health Organization (WHO), depending on hemoglobin (Hb) level, age and gender . The detThe preoperative identification and treatment of ID with or without anemia is a cornerstone of Patient Blood Management (PBM). In context of PBM, the diagnosis of ID and/or IDA in adults and in children is recommended by measurement of ferritin and transferrin saturation (TSAT) . The treOverall, IDA is detected relatively late by classic laboratory parameters such as Hb, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). A single biomarker is rarely used for the diagnosis of IDA or non-anemia iron deficiency (NAID), but the use of a marker that can be easily used as a screening marker is necessary. The lifespan of circulating erythrocytes is about 120 days. Changes in MCV and Hb values occur at a later time point when IDA is already fulminant . ReticulIn adults, the Ret-He has already been proposed as an additional marker to screen for ID ,14,15,16This retrospective study was approved by the Ethics Committee of the University Hospital Frankfurt, Goethe University, Germany (Ref: 19–338), and has been performed in accordance with the Declaration of Helsinki. The requirement for written informed consent by patients was waived by the Ethical Committee. This manuscript adheres to the applicable Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines.Between 1 January and 31 December 2019, hematological parameters  and serum iron parameters  and Ret-He) from children between >6 months and <18 years of age were retrospectively extracted from the hospital’s medical and laboratory records. Study population includes in-patient and out-patient children from any pediatric discipline. Laboratory examination was performed at any time during the in-hospital stay.In this study, anemia was defined according to the WHO definition for children that is irrespective of gender in children aged 6–59 months with a Hb level < 11.0 g/dL, children aged 5–11 years with Hb < 11.5 g/dL and children aged 12–14 years with Hb < 12.0 g/dL. For children at the age of ≥15 years, anemia was defined as Hb < 12.0 g/dL in female children and Hb < 13.0 g/dL in male children [Iron deficiency was defined by TSAT < 20% and serum ferritin level < 100 ng/mL, according to Munoz ,25, Anke6/µL)) × 10. MCV was calculated using the following formula: (Hb (g/dL)/red blood cell (×106/µL)) × 10. RCDW was calculated using the following formula: (1 SD/MCV) × 1000. The following parameters were measured by using Roche instruments ; serum iron (cobas 8000 c701); ferritin (cobas 8000 e801); and sTfR (cobas 8000 c502). Transferrin saturation was calculated as follows: (serum iron (mg/dL)/transferrin (mg/dL)) × 70.9 [All of the patients’ data were automatically extracted from the electronic laboratory information system, swisslab , version 2.18.1 until November 2019; swisslab II , version 2.21.6 from December 2019. Hemoglobin was measured using the XN-1000 . Ret-He was measured by fluorescence flow cytometry in the reticulocyte channel of the XN-1000 . MCV, MCH and RCDW were calculated automatically by the XN-1000 . MCH was calculated using the following formula: (hematocrit (%)/red blood cell (×10) × 70.9 .Primary endpoint was the validation of Ret-He as a screening marker for ID and IDA in children.Secondary endpoints were the children’s Ret-He levels in the four groups and correlation between Ret-He and ferritin or TSAT.According to Hb level and iron status, four groups were formed for preliminary analyses: the IDA group (anemia plus ID), NAID group , control group  and group of others . p-value of <0.05 was considered statistically significant.Data are presented as mean ± standard deviation (SD) or median and interquartile range (IQR) . Boxplots were used to illustrate data distribution. The Shapiro–Wilk test was performed to assess the normality of continuous variables. As all continuous variables were non-normally distributed, groups were compared by using the Kruskal–Wallis test. Then, a post hoc test (Dunn’s test) was applied to assess differences between the groups. Spearman’s correlation was used to investigate the correlation between Ret-He and ferritin or TSAT. A In addition, Receiver-operating characteristic (ROC) analysis was performed to assess the potential capabilities of Ret-He as a marker for ID (with and without anemia) and IDA, computing the ROC curve and its AUC ). The results of this analysis can be shown as the ROC curve, in which the sensitivity is plotted against the value of 1-specificity. If the AUC is almost 1, the accuracy of the test should be considered excellent. The objective is to find a cut-off value for Ret-He for ID and IDA in children as a screening parameter. Therefore, a sensitivity of 90% was defined as the threshold.®SPSS® Statistics .All statistical analyses and graphical illustrations were performed by using R©  and IBMBetween 1 January 2019 and 31 December 2019, data from 972 cases were identified. Of these, two cases were excluded due to incomplete medical records. In total, 970 cases were included in the final analysis. Overall, there were 506 (52.2%) male and 464 (47.8%) female children. The mean age was 11.2 (±4.9) years. There were 278 (28.7%) children in the IDA group, 332 (34.2%) children in the NAID group, 302 (31.1%) children in the control group and 58 (6.0%) children in the group of others. As expected, the median Hb level was the lowest in IDA children  g/dL) compared with NAID children  g/dL), children in the control group  g/dL) and children in the group of others  g/dL) . In Tablp < 0.001) or the control group  pg; p < 0.001). In addition, the differences in Ret-He values differed significantly between the NAID group and the control group (p < 0.001)  pg) compared to the NAID group  pg; < 0.001) .p < 0.001) (As presented in < 0.001) a.p < 0.001) (p < 0.001)  b. Furthe< 0.001) c.The diagnostic accuracy of Ret-He in screening for ID was assessed by ROC analysis. The AUC for Ret-He to screen for ID was 0.771. We calculated the sensitivity of Ret-He at a sensitivity of more than 90%. For a sensitivity of 90.7% and a specificity of 35.8%, the cut-off value of Ret-He was 33.5 pg .The AUC for Ret-He to screen for IDA was 0.845. Again, we calculated the sensitivity of Ret-He at a sensitivity of more than 90%. For a sensitivity of 90.6% and a specificity of 50.4%, the cut-off value of Ret-He was 31.6 pg .Therefore, the present data indicate that Ret-He may be an effective marker with sufficient sensitivity to screen for ID and IDA in children. This study evaluated the validity of Ret-He as a screening marker for ID and IDA in children between >6 months and <18 years of age. The analysis revealed that children with IDA had significantly lower Ret-He levels than children with NAID or children with normal iron and Hb values. In addition, Ret-He correlated positively with common laboratory parameters for ID, including ferritin and TSAT. As a result of the ROC analysis, this study shows the validity for using Ret-He to screen for ID (AUC = 0.771) and IDA (AUC = 0.845). Due to the retrospective study design of the present study, some disadvantages are obvious. However, the data were queried and evaluated according to standardized methods. It has to be mentioned that pathophysiological reasons and treatments causing states of ID and IDA were not analyzed. However, this study represents one of the largest pediatric patient populations at a PBM center in order to evaluate the usefulness of Ret-He as a screening marker for ID and IDA in children. The context of PBM screening for iron depleted states in children is especially crucial, as iron supplementation may be considered if indicated. In addition, any associated acute or chronic medical aspect was not considered in the present study. Therefore, children with hemoglobinopathies  were not identified and included in our analysis. In order to address the usefulness of Ret-He, especially in children with red blood cell disorders such as thalassemia, this particular patient cohort should be considered in future studies. A challenge are times of the blood draws and laboratory examinations during the children’s hospital stay, as they were not available and could not be correlated. Overall, parameters such as ferritin and TSAT are widely used in the context of ID. Nevertheless, recent studies have demonstrated that Ret-He may also be advantageous for the screening of ID, because it can be easily obtained and is free from biological variability ,32. Due p = 0.57). Serum ferritin levels differed between IDA and thalassemia (p < 0.0001). The authors suggest that upon diagnosis of iron depletion by Ret-He with potential diagnosis of thalassemia, serum ferritin should be determined. Especially in countries with high thalassemia prevalence, the usefulness of Ret-He to distinguish between IDA and hemoglobinopathy carriers has to be studied in future trials [In our study, the comparison of Ret-He levels between the patient groups revealed that children in the IDA group had significantly lower Ret-He levels than those in the control group. Moreover, data indicated that the decrease in RET-He was dependent on the severity of ID, which may lead to states of IDA. These results suggest that Ret-He reflects the iron state of children and may have potential as a useful screening marker for ID. Of note, Ret-He may also be low in hemoglobinopathies such as α-thalassemia or β-thalassemia. A retrospective analytical study by Chinudomwong et al. analyzed 253 children with IDA and 276 children with thalassemia. The results revealed that there was no significant difference in Ret-He values between children with IDA and thalassemia (median 20.6 pg (IQR) 9.0–19.6 pg; e trials . Furthermore, the comparison of Ret-He in all patients with standard parameters of iron metabolism (ferritin and TSAT) showed significant positive correlations of Ret-He with ferritin and TSAT. These results are in line with a previously published study of 100 children suffering from cancer. The authors also detected a correlation between Ret-He and ferritin in a subgroup analysis in patients with ID . AnalysiIn this study, ROC analysis revealed a valid utility of Ret-He in screening for ID (AUC = 0.771) and IDA (AUC = 0.845). For pediatric patient groups, cut-off values between 26.0 pg and 29.0 pg have been reported with varying sensitivity and specificity  20,21,2,34. In ap < 0.001) and 4 weeks (p < 0.001) strongly correlated with response to intravenous iron supplementation compared to baseline values [Iron deficiency anemia can be treated with intravenous or oral iron supplementation. A study on 65 hemodialysis patients with IDA and intravenous iron administration revealed that changes in Ret-He during iron treatment after two weeks  may be of high interest in future studies. Economically, the use of Ret-He in screening for ID and IDA is highly relevant as it can save one blood tube per patient and additional costs.


Background and objectiveSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China in December 2019. Since then, It has spread across multiple countries and was declared a pandemic by WHO in March 2020. Patients with underlying diabetes mellitus (DM) are deemed at-risk for developing severe COVID-19 infection. In light of this, we aimed to evaluate the correlation between DM and chest CT severity scores (CTSS) in COVID-19 patients.MethodsThis was a hospital-based descriptive, analytical retrospective study conducted at our tertiary care hospital. A quantitative severity score was calculated among 220 patients with COVID-19 infection based on the degree of lung lobe involvement on CT chest scans. Based on CTSS, the patients were classified into groups of mild, moderate, and severe lung involvement. The association between DM and CTSS was evaluated using the chi-square test.ResultsThe severity of lung involvement was higher among COVID-19 patients with a co-diagnosis of DM (29.3%) compared to those without DM (11.7%). This association of severe lung involvement with DM was statistically significant (p=0.002).ConclusionBased on our findings, diabetic patients are at an increased risk of developing the severe form of COVID-19 with a higher CT lung involvement score compared to non-diabetic patients. In December 2019, the first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in the city of Wuhan in China. By January 2020, the virus had rapidly spread across the Wuhan province as well as other provinces in China [Due to the disease's novelty, the criteria that influence the severity of the condition and mortality remain largely unclear. Patients with underlying health problems, people over 65 years of age, and delayed hospitalizations are all considered to play a role in the severity of the symptoms -5. PatieA worldwide prevalence of diabetes mellitus (DM) has affected approximately 463 million people . As of 2The goal of this research was to fill the gaps in the existing literature by conducting a study that analyzed current data to determine the extent of the association between COVID-19 severity and DM. We hoped that our findings would contribute toward complication prevention and better patient management stratification.Study designWe conducted a hospital-based descriptive and analytical retrospective study at our tertiary care hospital after obtaining approval from the Institutional Ethics Sub-Committee (IESC) of Dr. D. Y. Patil Medical College, Hospital & Research Centre, Pune, IND. The approval number assigned to our study is IESC/FP/2021/53.Inclusion criteriaPatients of both genders and all age groups who had undergone high-resolution CT (HRCT) of the lung after testing positive for COVID-19 infection on reverse transcription-polymerase chain reaction (RT-PCR) were included in our study.Exclusion criteriaPatients whose CT could not be evaluated due to severe motion artifacts or other technical issues such as restricted field of view or those with CT studies requiring contrast medium were excluded from our study.Data collectionThe results of RT-PCR were considered confirmatory for the diagnosis of COVID-19 infection. The HRCT scans were performed between the fifth and 10th days after the onset of the symptoms. HRCT of the lung was performed on a multidetector 128-slice CT scanner . Patients were placed in the supine position and asked to hold their breath at the end inspiration for non-contrast-enhanced HRCT scans. The following technical parameters were used: tube voltage of 120 kV, tube current of 100-200 mAs, collimation of 1.5-3 mm, and slice thickness of 0.625-1.25 mm. The high-spatial-frequency (bone) reconstruction algorithm was used to evaluate the lungs. The data from the HRCT lung studies of 220 patients performed between March and May 2021, i.e., during the second COVID-19 wave, was retrospectively collected and analyzed to determine the association between diabetes and COVID-19 severity. All the scans were assessed by a senior radiologist with 15 years of work experience to avoid inter-observer variation in the severity scoring of the COVID-19 lung infection. A quantitative CT score was calculated based on the degree of lung lobe involvement  . Based oStatistical analysisStatistical analysis was performed by entering the data into Microsoft Excel and evaluating it using the SPSS Statistics software version 17.0 . Patients’ gender, the status of DM, BMI, as well as ICU admissions were reported as percentages. The CT score was categorized into mild (0-11), moderate (12-18), and severe (>18) categories. Then mild and moderate groups were combined for the purpose of analysis. The association between DM and severe COVID-19 was calculated using the chi-squared test. A p-value <0.05 was considered statistically significant.For the purpose of the present study, we retrospectively reviewed HRCT scans and the clinical data of 220 patients. Out of those 220 patients, 136 (61.8%) were males and 84 (38%) were females, with a mean age of 48.69 ±16.07 years  had a milder form of lung involvement as per the CTSS category; 70 patients (31.8%) had a score of 12-18, which corresponded to moderate involvement, and the remaining 32 (14.6%) patients had a severe form of lung involvement  . The casIt is unknown how diabetes worsens COVID-19, but numerous variables might be at play . InadequThe relationship between COVID-19 and diabetes might be bidirectional, with SARS-CoV-2 possibly exacerbating underlying diabetes or perhaps predisposing non-diabetic individuals to diabetes. SARS-CoV-2 enters human cells through the ACE2 pathway, and ACE2 is widely expressed in the liver and endocrine pancreas, perhaps contributing to insulin resistance and reduced insulin production . In suscThe present study has shown that diabetic patients are at an increased risk of developing the severe form of COVID-19 with a higher CT lung involvement score than non-diabetic patients. Thus, it is essential to ensure strict glucose monitoring in COVID-19 patients with DM to prevent the occurrence of life-threatening complications. Diabetes should be regarded as a risk factor for the severity of COVID-19 disease, and minimizing exposure to sources of COVID-19 is the best possible method to reduce morbidity and mortality.


Background: Obesity is an important pathology in public health worldwide. Obese patients are characterized by higher cardiovascular risk and a pro-inflammatory profile. Objective: To assess the oxidative stress in peripheral blood mononuclear cells (PBMCs) and inflammatory biomarkers in plasma in adults with normal weight, overweight and obesity. Methods: One hundred and fifty adults  from the Balearic Islands, Spain, were recruited and classified according to body mass index (BMI). Anthropometric measurements were carried out, fasting blood samples were collected and plasma and PBMCs were obtained. Biochemical parameters, hemogram, antioxidant enzyme activities and protein levels, reactive oxygen species production (ROS), malondialdehyde (MDA), and cytokine  levels were measured. Results: Glycaemia, triglyceridemia, abdominal obesity, and waist-to-height ratio (WHtR) were higher, and HDL-cholesterol was lower in obese patients. MDA and TNFα plasma levels were higher in the obese compared to normal-weight group, while the levels of IL-6 were higher in both obese and overweight subjects with respect to normal-weight peers. The activities of all antioxidant enzymes in PBMCs as well as the production ROS progressively increased with BMI. The protein levels of catalase in PBMCs were higher in obese and glutathione reductase in obese and overweight subjects compared to normal-weight peers. No other differences were observed. Conclusion: The current results show that overweight and obesity are related to an increase in pro-oxidant and proinflammatory status in plasma and PBMCs. The studied biomarkers may be useful for monitoring the progression/reversal of obesity. Obesity is considered a clear threat to public health worldwide, and, together with overweight, constitutes one of the main factors that promote non-communicable diseases (NCDs) . Direct Obesity is characterized by an increase in oxidative stress and a pro-inflammatory state . The patA possible cause of the obesity-induced pro-inflammatory state, based on the establishment of oxidative stress, is a high concentration of pro-inflammatory cytokines in serum in these patients . TNF-α aPeripheral blood mononuclear cells (PBMCs), comprising lymphocytes and monocytes, are cells that play crucial roles in the immune system, and they also participate in pathological processes associated with obesity or metabolic syndrome (MetS) ,11. AccoOne hundred and fifty participants  who were residents of the Balearic Islands were included in the study. This sex-age range was chosen because people are at high risk of overweight/obesity and related co-morbidities  at this age.2), overweight (25.0–29.9 kg/m2) and obese (more than 30.0 kg/m2) [The participants were distributed according to their body mass index (BMI): normal weight (18.5–24.9 kg/m0 kg/m2) . Patient2). A validated semi-automatic oscillometer  was used to measure blood pressure; measurements were made in the arm, registering the highest diastolic blood pressure, taking three measures after the participant was seated for 5 min, waiting for 1 min between each determination. An anthropometric tape was used to measure abdominal obesity, halfway between the last rib and the iliac crest. Waist-to-height ratio (WHtR) was calculated as waist (cm)/height (cm). The physical activities performed were evaluated as metabolic equivalents (METs) considering the rate of energy waste [To reduce the inter-observer coefficients of variation, the anthropometric variables were carried out by specialist observers. Height was determined with an anthropometer  to the closest millimetre. A Segmental Body Composition Analyzer  was used to determine body weight and body fat, with subjects in light clothes and without shoes, and subtracting 0.6 kg for the clothes. BMI values were calculated as weight (kg)/height . The final precipitate of PBMCs was washed with phosphate-buffered saline (PBS) at pH 7.4 and centrifuged at 900× g, for 10 min at 4 °C. Venous blood samples from all participants were obtained after a 12 h night-time fast in ethylene diamine tetraacetic acid (EDTA) sample tubes and were centrifuged at 1700× od cells  was usedGlucose, triglycerides, high-density lipoprotein (HDL) and total cholesterol were determined in plasma by standard procedures using standard enzymatic methods. Hemogram and haematological parameters were measured using an automatic flow cytometer analyser Technicon H2 . TM (Bio-Rad) as a molecular weight marker. The electrophoresis was at 60 V for 15 min and then 150 V for 1 h. A Trans-Blot® TurboTM Transfer System  was used to electro-transfer the obtained bands onto a 0.2μm nitrocellulose membrane. After blocking the membranes with 5% non-fat powdered milk in TBS pH 7.5, 0.1% Tween 20, they were incubated overnight at 4 °C with the primary monoclonal antibodies. Antibodies anti-catalase (CAT) ref. 219010  and anti-Mn superoxide dismutase (MnSOD) ref. 574596  ; anti-glutathione (GPx) ref. sc-22146 , anti-glutathione reductase (GRd) ref. sc-133136  and actin ref. sc-8432, used as the loading control  . A secondary peroxidase-conjugated antibody against specific primary antibody, for anti-CAT (ref. A0545) (1:5000), anti-MnSOD (ref. A3415) (1:5000) and anti-GRd (ref. A9044) (1:5000) and anti-GPx (ref. A5420) (1:10000) was used. Immunoblots were revealed by an enhanced chemiluminescence kit  to visualize the antibody-bound proteins. Signals were detected using a ChemiDoc XRS charge-coupled device imaging camera and quantified using the image analysis program Quantity One (Bio-Rad). Chemiluminescence exposure times to a film/CCD camera were varied over a period of 5–500 s to better resolve the different protein bands. Actin was used as a housekeeper protein to normalize Western blot bands for protein loading, since its expression was unaffected by the experimental treatments. It is considered a normal weight group with 100% of protein levels; therefore, the overweight and obese groups were recalculated with respect to it.Western blot analysis was used to assess the levels of the PBMCs’ antioxidant enzyme proteins of all participants. In this process, 50 µg of protein from lysed cells was placed in an 15% SDS-polyacrylamide gel using Precision Plus Protein Kaleidoscope2O2 [−1)/109 cells and the other enzymatic activities as nkatal (nKat)/109 cells. All the enzymatic determinations in PBMCs were determined using a Shimadzu UV-2100 spectrophotometer  at 37 °C. Catalase (CAT) activity was analysed by the spectrophotometric method of Aebi based on the decomposition of H2O2 . Superox2O2 . Glutath2O2 . Glutath2O2 . The act®, St. Louis, MO, USA) following the manufacturer’s instructions [A marker of lipid peroxidation, malondialdehyde (MDA) concentration (nM), was analysed in plasma and in PBMCs of all participants by a specific colorimetric assay kit . Cell suspensions (6 × 105 cells) were introduced in a 96-well microplate and LPS prepared in 2 mM in phosphate buffer saline, pH 7.4 was also added to the wells. Then, the cell-permeant probe 2,7-dichlorofluorescein-diacetate  was added to all wells. The fluorescence  was recorded at 37 °C for 60 min in FLx800 Microplate Fluorescence Reader .PBMCs ROS production was monitored after activation with lipopolysaccharide (LPS) from Cytokine (TNFα and IL-6) levels were determined in plasma of all participants using individual ELISA kits  following the manufacturer’s instructions for use. The intra- and inter-assay coefficients of variation were 3.2% and 10.9% for TNFα and 4.4% and 9.1% for IL-6, respectively. p < 0.05 as statistically significant. Kolmogorov–Smirnov test was used to assess normality of data. One-way analysis of variance (ANOVA) after adjustment for BMI was used to check significance of data. Bonferroni post hoc test was carried out when significant differences were found between groups. A receiver operating characteristic (ROC) curve and area under the curve (AUC) was used to analyse the biomarker results.Statistical analysis was carried out with the Statistical Package for Social Sciences . Continuous variables were presented as means ± standard error of the mean (SEM), and considering Haematological parameters of participants are presented in The activities of the antioxidant enzymes in PBMCs are shown in Plasma and PBMCs MDA levels, a marker of lipid peroxidation, were shown in The ROS production by PBMCs stimulated with LPS is presented in The plasma proinflammatory cytokine concentration is presented in The main findings of the current study are the progressive increase in the number of circulating leukocytes, and the degree of oxidative stress and pro-inflammatory biomarkers in PBMCs and plasma, which occurs with overweight and obesity. The higher number of leukocytes, neutrophils, basophils, and monocytes observed in obese participants when compared with normal-weight peers is probably related to the proinflammatory state associated with this disorder. Previous studies suggested that the mobilization of immune cells and the release of proinflammatory mediators can be caused by the inflammation of adipose tissue due to overweight and obesity ,22. SpecObese subjects from the present study also showed higher plasma levels of the pro-inflammatory TNFα and IL-6 with respect to normal-weight peers. Obese subjects have a persistent low-grade inflammatory state which alters adipocyte metabolism and gene expression, increasing lipolysis and the release of mediators that recruit and activate macrophages such as TNFα, IL-6, IL-1β and monocyte chemotactic protein-1 (MCP-1) ,24. ThesMoreover, current participants also showed a progressive increase in abdominal obesity and WHtR that could be responsible for the increase in circulating levels of both pro-inflammatory cytokines. The present results confirm that obese participants had chronic inflammation due to an increase in circulating inflammatory cytokines that can contribute to mobilize and pre-activate the immune cells, mainly neutrophils and monocytes. In addition, higher plasma levels of MDA were also found in obese subjects than in normal-weight and overweight participants. MDA is a widely used marker of oxidative damage as well as the main end-product of lipid peroxidation . The obt2O2 induces the expression of Toll-like receptors (TLRs) 2 and 4 that activate pro-inflammatory signalling pathways such as the nuclear factor κB pathway (NFκB) pathway [2O2 (produced with glucose oxidase) in PBMCs from patients with metabolic syndrome to mimic chronic stress caused a transient inflammatory response with an increase in the production of IL6, IL8, and IL1β [The increased activity and protein levels of antioxidant enzymes in PBMCs observed in overweight and obese subjects from the present study may be a consequence of a pre-activation state of these cells, which is a derivative of chronic inflammation and oxidative stress-related to excessive fat accumulation ,34. In t pathway . In addi pathway ,40. Thus pathway ,42. Incr pathway . Accordiand IL1β . The obsand IL1β . On the and IL1β . Moreoveand IL1β . As was expected, current obese patients also showed a higher body weight, BMI, abdominal obesity, WHtR, glucose and triglyceride levels, and lesser HDL-cholesterol than normal-weight patients, in accordance with previous studies reporting that an increase in visceral abdominal fat volume is proportionally correlated with an increase in WHtR and BMI in overweight and obesity, and these patients have a major incremental on the cardio-metabolic risk factors ,7.The present results from the ROC curve and area under the curve show that the PBMCs’ levels of SOD, GRd, CAT, IL6, and TNFα may be good BMI markers with potential utility in clinical practice.The main strength of this study is that an association has been found between plasma and PBMC oxidative stress and proinflammatory biomarker levels and normal weight, overweight, and obese stages. The main limitation of this study is that sample size was relatively reduced; nevertheless, the sample size was enough to evidence the existence of differences in the biomarker levels between normal-weight, overweight, and obese participants in the study.The current study has demonstrated a progressive increase in oxidative stress and inflammation among overweight and obese individuals when compared with normal-weight subjects. Plasma oxidative stress and inflammation biomarkers were mainly increased in obese subjects. Nevertheless, in PBMCs, the antioxidant defences were seen to increase as a compensatory mechanism in the face of a chronic pro-oxidative and pro-inflammatory stimulus situation. The relationship between the biomarkers studied and overweight/obesity suggests that they may be useful for monitoring the progression/reversal of obesity.


Patients and Methods. Consecutive patients undergoing colonoscopy were randomized (1 : 1) into examination by ME-OE or ME-CE. Histopathological findings were utilized as the reference standard. Accuracy, sensitivity, specificity, and positive and negative predictive values of two endoscopy methods were compared using ME-OE (were classified according to the JNET classification) and ME-CE (were classified according to the Kudo pit pattern classification), respectively, and the time to predict the histological polyp type was compared. And the agreements between the pathological and clinical diagnosis by ME-OE or ME-CE were analyzed.  Magnifying chromoendoscopy (ME-CE) through the observation of pit patterns is a productive way to distinguish between neoplastic and nonneoplastic polyps. Magnifying optical enhancement technology (ME-OE) is an emerging virtual chromoendoscopy imaging technology and appeared to be a promising approach. However, this information is currently not available. This study is aimed at comparing the differential diagnostic value of ME-CE and OE for neoplastic and nonneoplastic polyps. p > 0.05). The average diagnosis time was lower in ME-OE than ME-CE . The agreements between the pathological and clinical diagnosis were at least substantial in both groups.  A total of 365 polyps were found in the 220 patients included . The diagnostic accuracy of ME-OE was higher than that of ME-CE (93% vs. 92%, ChiCT2000032075. ME-OE was superlative to ME-CE in predicting the histology of polyps. OE devoted classification would possibly similarly enhance the endoscopist performance. The trial is registered with  In 2018, an additional 1.8 million new cases of colorectal cancer were diagnosed, with 881,000 deaths. Colorectal cancer accounts for one in 10 cancer cases and deaths, the 3rd incidence and the 2nd mortality . MultiplFurthermore, since diminutive polyps account for the majority of polyps found during the colonoscopy, the ability to predict polyp histology in real time is clinically significant . The JNESince the 1970s, Japanese endoscopists have used chromoendoscopy, and the Kudo proposed pit pattern classification has been generally adopted. Using the Kudo pit pattern classification system, chromoendoscopy with or without magnification has been used to discern neoplastic from nonneoplastic polyps . The optChiCT2000032075).This was a prospective, randomized, and single-center study conducted at a teaching hospital in Zhengzhou, China. All participating patients provided written informed consent. The Ethics Committee approved Zhengzhou Central Hospital Affiliated to Zhengzhou University's research protocol (202032), and the study is registered in the China Clinical Trial Registry  assessment. The exclusion criteria were as follows: inadequate bowel preparation defined as a total BBPS score < 6 , previouAll procedures were performed with an EPK-i7000 processor , high-definition endoscopes with magnification (EC-3890FZi), and a black silicone elastomer cap on the distal end of the colonoscopy . All patients received a low-residue diet two days before the colonoscopy, oral polyethylene glycol cathartic agent 3L, to make the bowel preparation meet the requirements. All patients were given nasal cannula oxygen during the anesthesia process, a certified anesthetist performed the entire anesthesia process, and vital signs were monitored throughout the procedure. In our study, antispastic agents were not provided.A complete examination was defined as reaching the caecum using WLE. The effectiveness of the bowel cleansing was assessed according to the Boston Bowel Preparation Scale (BBPS) at the withdrawal phase. When a macroscopically visible lesion was suspected by white light endoscopy, the polyps' location sizes and morphology were also recorded. To expose the microstructure and microvasculature on the surface of the polyp, pronase and simethicone were applied. The diagnosis of polyp histology was based on diagnostic criteria defined by JNET or Kudo classification. JNET type 1 was classified as nonneoplastic polyps; other types were considered neoplastic polyps ; pit patThe study flowchart is shown in The tissue specimens collected during endoscopies were placed into formalin solution for 24 hours, subjected to conventional dehydration, paraffin embedding, sectioning, and then staining using the hematoxylin-eosin (H/E) staining method. Two experienced pathologists reviewed the slides independently and reached histological conclusions without knowing the endoscopic findings. If the results of the two pathologists were notably different, a third pathologist was consulted. The diagnostic histopathological criteria were based on the Vienna classification .With histopathological evaluation as a reference standard, we evaluated the accuracy, sensitivity, specificity, and positive and negative predictive values of ME-OE and ME-CE endoscopy for polyp histology prediction, as primary outcomes.The secondary outcome measures included comparing the time needed to predict polyp histology and the agreements between the pathological and clinical diagnosis by ME-OE or ME-CE.β, is set to be 80%. With 10% expected dropouts, the total sample size was 220, who prudentially assume a detection of only one polyp. Combined with my endoscopic center, the detection rate of polyps is about 35%. We planned to enroll 628 patients.The overall diagnostic accuracy in differentiating neoplastic from nonneoplastic lesions in their series was 80% . We havet-test. Consistency analysis was evaluated by the kappa test. The larger the kappa, the higher the agreement.Statistical analysis was performed using SPSS 26.00 statistical software. Frequencies with percentages were used to represent qualitative variables, whereas means and standard deviations were used to describe quantitative variables. Comparisons of qualitative variables were conducted using Fisher's exact probability test or chi-squared test, while for comparisons of continuous variables, we employed a From August 2019 to May 2020, we screened 519 patients, and after, 229 patients were excluded . A totalOur main targets here are to compare the accuracy of polyp diagnosis between groups.p > 0.05. The accuracy of ME-OE in stage 1 and 2 was 90% vs. 96%, p > 0.05. The accuracy of ME-CE in stages 1 and 2 was 90% vs. 93%, p > 0.05 , 96 s ± 26.8 s and 70 s ± 19.0 s (p < 0.001) in two stages of ME-OE, and 20.5 s ± 13.9 s and 183 s ± 14.0 s (p < 0.001) in two stages of ME-CE  in stage 1 and 70 s ± 19.0 s for ME-OE and 183 s ± 14.0 s for ME-CE (p < 0.001) in stage 2 (The average diagnosis time between the various groups in the same stages was compared: 96 s ± 26.8 s for ME-OE and 205 s ± 13.9 s for ME-CE ( stage 2 .k = 0.859) and ME-CE (k = 0.827), both almost perfect (k = 0.804 and 0.912) in 2 stages of ME-OE, and substantial (k = 0.800) for stage 1 and nearly perfect (k = 0.848) for stage 2 in ME-CE . In stage 1, the overall accuracies were 90% and 90% for ME-OE and ME-CE, p > 0.05. In stage 2, the overall accuracies were 96% for ME-OE and 93% for ME-CE, p > 0.05. Whether from the overall accuracy or the same group of different stages, the same stage of the other groups' accuracy is more than 90% accurate. The agreements between the pathological and clinical diagnosis are healthy at all times. It can be seen that both groups have effective methods to diagnose the nature of polyps. This point needs attention. The performance of ME-OE in classifying colorectal polyps is much better than ME-CE at all times. Especially in OE stage two, the specificity was 100%, and there were no cases with misdiagnosis. While the ME-CE is also excellent, the ME-OE seems to be the better choice.Compared to the accuracy of the two groups, the diagnostic accuracy of ME-OE was significantly higher than that of ME-CE . This superiority continues until the time point before the last. This may be related to the operation process of pigment endoscopy, which requires not only delivering the spray tube to the intestinal cavity and fixing the spray tube to a specific position to spray accurately but also removing the excess solution. Besides, there are differences between different stages within the same group. In both groups, the second stage was faster than the first. This might be due to a lack of experience in the early. In a study of endoscopists with different experiences, after 20 minutes of NBI teaching , it is sThis study also had some limitations. Namely, this was a single-center study with a small sample size, and thus, further in-depth, large-scale, and multicenter studies are required in the future. Besides, no specific classifying colorectal polyps for ME-OE are available thus far, and we hope that a ME-OE diagnostic standard can be established shortly. The examination was performed by experienced endoscopists, which may lead to selection bias. As the limitation of the experimental specimens, we did not collect all the polyp types, which may have an individual impact on the conclusion.Overall, the study adds to our understanding of classifying colorectal polyps by ME-OE. And the ability of ME-OE was significantly better than that of ME-CE. The method not only has high accuracy but also is simple to perform with ME-CE and saves time. Furthermore, practical learning is necessary to raise diagnostic accuracy. In the future, we should further verify the ability to use OE to identify various polyp types based on JNET classification.